We plan to continue studies on in vitro synthesis of RNA and DNA. In studies on DNA replication we will investigate reactions involved in converting phi X single-stranded circular DNA to RFII. This system has been shown to involve 10 proteins, each of which has been isolated free of the others. The omission of any one protein involved in this reaction results in no detectable DNA synthesis. This reaction has been dissected into at least two steps. The first step involves dna B, dna C (D), DNA binding protein, replication factors X, Y and Z, ATP and phi X DNA. The product of this reaction can be isolated by gel filtration and shown to lack detectable dna C (D) and replication factor X; this product when supplemented with dna G, Pol III, elongation Factor I and II and the 4 dXTPs, supports DNA synthesis. We plan to 1) determine the role of ATP in the reactions, 2) elucidate the mechanism involved in DNA initiation and 3) define the biochemical role of each of the proteins. Our studies on RNA synthesis will focus on the requirements for transcription of chromatin using eucaryotic RNA polymerase II (B). We plan to examine the nature of the chromatin template which can lead to RNA products containing biologically defined regions (globin, AMV, FLV). We have already utilized chromatin from myeloblasts of AMV-infected birds; transcription of such chromatin in vitro has yielded RNA containing AMV sequences.